WO2011134098A1 - Method for inhibiting tau protein aggregation and treatment of alzheimer's disease with a compound derived from quinoline - Google Patents

Abstract

Method for inhibiting tau protein aggregation and treatment of Alzheimer's disease, administering a derivative of quinoline having formula (I), where R₂ is 2-(4-aminophenyl) or 2-(4-methylphenyl) and R₆ is methyl, as inhibitor of tau protein aggregation.

Description

 METHOD FOR INHIBITING THE ADDING OF TAU PROTEIN AND TREATMENT OF ALZHEIMER'S DISEASE. WITH A COMPOUND DERIVED FROM QUINOLINA. FIELD OF THE INVENTION

 The present invention relates to specific quinoline molecules, polymerized tau binding ligands, as potential blockers of tau aggregation before the formation of NFTs. Said quinolines preferably have an amino group or a methyl group at the meta position of the nitrogenous ring, and which show the highest disaggregation activity of tau polymers, and are therefore useful in the treatment of Alzheimer's disease. The present invention provides quinoline compounds that inhibit formation and at the same time disaggregate NFTs, and therefore, would be useful in the treatment and prophylaxis of Alzheimer's disease.

PRIOR ART

Alzheimer's Disease (AD) is a neurodegenerative disease, of slow evolution and the most common cause of dementia, mainly affecting 2% of the population under 65 years and almost 50% older than 85 years. So it is imperative to have a treatment, and thus avoid an eventual and serious epidemiological process that could be triggered within the next decade for not having such treatment (Green RC, Cupples LA, Go R., Benke KS, Edekí T., Griffith PA, Williams M., Hipps Y., Graff-Radford N., Bachman D., Farrer LA (2002). MIRAGE Study Group. Risk of dementia among white and African American relatives of patients with Alzheimer disease. ., 287 (3): 329-36; Maccioni RB, Lavados M., Maccioni CB and Mendoza A. (2004). Biological markers of Alzheimer's disease and mild cognitive impairment. Current Alzheimer Research. 1: 307-314; Maccioni RB Farias GA Red LE, Sekler Kuljis MA and RO (2008) What have we Learned from the tau hypothesis in:... Hypotheses and Research Milestones in Alzheimer ^'s Disease (RB & G. Perry Maccioni, eds.) Springer. -Verlag, New York-Heidelberg). AD is also one of the biggest public health problems because it is one of the diseases that has the most economic impact on modern society (Wimp A., Winblad B. (2001). Health economical aspects of Alzheimer disease and ts) treatment. Psychogeriatrics. 1: 189-93; Winblad B. (2001). Maintaining functional and behavioral abilities ¡n Alzheimer disease. Alzheimer Dis Assoc Disord. 1: S34-40). It is considered as a clinical-pathological entity consisting of the association of a slowly progressive dementia, beginning in adulthood, and the observation in the cortex of neuronal loss and presence of senile or neuritic plaques (PS) and neurofibrillar clews (NFTs) ), in concentrations that clearly exceed what is expected by an effect of physiological aging (Maccioni, RB, Barbeito L, and Muñoz JP (2001). The molecular bases of Alzheimer's disease and other neurodegenerative disorders. Arch. Medical Research. 32: 367- 381; Rojo LE., Fernández JA, Maccioni AA, Jiménez JM, Maccioni RB (2008). Neuroinflammation: implications for the pathogenesis and molecular diagnosis of Alzheimer's disease. Arch Med Res. 39 (1): 1-16). As a result of the degeneration of synapses and neuronal death, regions of the brain such as the frontal and temporal lobes, the latter involved in learning and memory processes, are affected and have a reduced size in patients with AD as clearly shown in Mattson M. (2004). Pathways towards and away from Alzheimer disease. Nature 430 (7000): 631-9. Because there are other types of dementias of different etiologies such as those of vascular origin, due to vitamin B 2 deficiency, neurodegenerative (such as frontotemporal dementia) and infectious (including syphilis, HIV-associated dementia and Creutzfeld-Jakob) , among others (Me Khann GM (1991). The future of child neurology, J. Child Neurol. 6 (2): 167-72), the differential and certain diagnosis of AD still requires a post-mortem neuropathological examination (Kuljis RO, Darvesh S., Greig NH, Geula C. (2006). Tomographic visualization of cholinesterase. Ann. Neurol. 60 (6): 745-6). This consists of histologically analyzing brain tissue sections that must have a sufficient number of PS and NFTs coexisting in certain areas of the brain such as the hippocampus and Meynert nuclei. The diagnosis is based on clinical criteria and neuropsychological examinations, identification of typical symptoms of the disease and the exclusion of other causes of dementia (Dubois B., Feldman H., Jacova C. (2007). Research criteria for the diagnosis of Alzheimer's disease : revising the NINCDS- ADRDA criteria. Lancet Neurol. 6: 734-46). In this sense it should be noted that the lack of A highly reliable biomarker of certainty has delayed investigations of AD on the one hand, and made the early diagnosis of this pathology more difficult.

 Clinically, AD is characterized by progressive deterioration and a decrease in cognitive functions, such as memory language and visual space orientation. AD is also characterized by a gradual loss of memory, decreased ability to perform routine tasks, spatial and temporal disorientation, learning difficulties, loss of linguistic ability, deterioration of reasoning, rapid changes in state of mood and personality disorders (Katzman R. (2004). Luigi Amaducci memorial award winner's paper 2003. A neurologist's view of Alzheimer's disease and dementia. Int Psychogeriatr. 16 (3): 259-73). In its initial phase, a loss of neurons is not observed, but rather a dysfunction of these, which gradually increases to more severe states in which a severe cognitive deterioration associated with neuronal loss in areas of the hippocampus, entorhinal cortex and then in the prefrontal and temporal cortex as clearly shown in Mattson M. (2004), Pathways towards and away from Alzheimer disease, Nature, 430 (7000): 631-9.

AD is related to a wide and gradual neuronal loss, but the main neuropathological event consists in the deposition of PS and NTFs. The PS are mainly formed by the variant (β (1-42) (Mattson M. (2004). Pathways towards and away from Alzheimer disease. Nature. 430 (7000): 631-9). In the case of NFTs, they consist of a protein associated with the neuronal cytoskeleton called tau, which is hyperphosphorylated in the brains of patients with AD. (Kurt MA, Davies DC, Kidd M. (1997). Paired helical filament morphology varíes with intracellular location in Alzheimer's disease brain. Neuros Lett. 239 (1): 41-4; Maccioni, RB, Barbeito L., and Muñoz JP (2001) .The molecular bases of Alzheimer ^' s disease and other neurodegenerative disorders. Arch. Medical Research. 32: 367-381; Maccioni RB, Lavados M., Maccioni CB and Mendoza A. (2004). Biological markers of Alzheimer ^' s disease and mild cognitive impairment. Current Alzheimer Research. 1: 307-314). Through unknown mechanisms, tau undergoes significant modifications, such as abnormal phosphorylation due to activity deregulated of various kinases and phosphatases which affect their normal biological function (Zambrano CA, Egaña JT, Núñez MT, Maccioni RB, González-Billault C. (2004). Oxidative stress promotes tau dephosphorylation in neuronal cells: the roles of cdk5 and PP1 Free Radie Biol Med. 36 (11): 1393-402). Under these circumstances tau begins to be added originating the NTFs, structures that constitute a characteristic histopathological marker of AD along with the PS (Maccioni C, Arzola ME, Mujica L. and Maccioni RB (2003). New paradigms in the study of pathogenesis of Alzheimer's disease Rev Chil Neuro-Psychiatrist 41 (2): 33-46). PS and NTFs are present mainly in regions of the brain involved in learning, memory and emotional behaviors, such as the hippocampus, cerebral cortex and the amygdala.

As for its pathogenesis, it is clear that the characteristic lesions of AD, that is, NFTs and senile plaques (PS), are not the triggering events of its pathogenesis, but the late result of processes that occur over many years. Different theories about AD have been postulated, but recently the unifying hypothesis of AD has found greater acceptance (Fernández JA, Rojo L, Kuljis R. and Maccioni RB (2008). "The damage signifies hypothesis of Alzheimer's disease pathogenesis", J. Alzheimer Dis. 14: 329-33; Maccioni RB, Rojo L, Fernández J. and Kuljis RO (2008). "Neuroimmunomodulation in Alzheimer ^' s disease." Ann NY Acad. Sci.), Which states that there is a series of damage-alarm signals in the innate immunity system, in the early stages of the pathogenesis of AD. Signs of endogenous damage such as Αβ oligomers, LDL oxidase, free radicals, mechanical damage and advanced glycosylation products (AGEs), caused by external factors such as trauma, high fat intake, vitamin B deficiency, infections and iron overload among others, they activate the microglia through AGEs receptors. Separately LDL-oxidases activate toll-like receptors (TLRs), in particular TLR4. Additional signs of damage such as trauma and free radicals possibly act on separate receptors as described in Fernández JA, Rojo L., Kuljis R. and Maccioni RB (2008). "The damage signifies hypothesis of Alzheimer's disease pathogenesis", J. Alzheimer Dis. 14: 329-33; Maccioni RB, Farias GA, Red LE, Sekler MA and Kuljis RO (2008). What have we learned from the tau hypothesis. In: Hypotheses and Research Milestones n Alzheimer ^'s Disease (RB & G. Perry Maccioni, Eds.). Springer-Verlag, New York-Heidelberg. Separately and in various combinations, these signals could trigger alarm mechanisms in the innate immunity system, resulting in an increase in NFK-B levels, a transcription factor that would increase the expression of proinflammatory cytokine genes (TNF-a, IL-Ι β, IL-6) released by microglia and promoting erroneous signaling cascades in the affected neurons (Rojo LE, Fernández JA, Maccioni AA, Jiménez JM, Maccioni RB (2008). Neuroinflammation: implications for the pathogenesis and molecular diagnosis of Alzheimer's disease Arch. Med. Res. 39 (1): 1-16). Thus, the levels of these cytokines are increased in the spinal fluid (CSF). These signals would be directly related to neuronal damage, because cell cycle enzymes such as cdk-5 would be activated, which is reflected in alterations such as tau protein hyperphosphorylation and paired helical filament formation (PHFs) , processes that result in neuronal degeneration and progressively severe clinical manifestations that affect cognitive and behavioral processes (Fernández JA, Rojo L, Kuljis R. and Maccioni RB (2008). "The damage signifies hypothesis of Alzheimer's disease pathogenesis", J. Alzheimer Dis. 14: 329-33).

 Senile plaques (PS) are structures located in the extracellular space where nerve endings move and correspond to deposits of amorphous fibrils and aggregates of β-amyloid (Αβ) (Liu WK, Ksiezak-Reding H., Yen SH (1991) Abnormal tau proteins from Alzheimer's disease brains. Purification and amino acid analysis. J Biol Chem. 266 (32): 21723-7). These are annular conglomerates of degenerated neuronal bodies and extensions around a central deposit of the Αβ peptide, which has a variable length of 39-43 amino acids. This peptide is derived from the proteolytic processing of the amyloid precursor protein (APP) (Glenner GG, Wong CW, Quaranta V., Eanes ED (1984). The amyloid deposits in Alzheimer's disease: their nature and pathogenesis. Appl Pathol. 2 ( 6): 357-69).

Three enzymes are responsible for this process. The APP can be fragmented by joint action of α-secretase, followed by the action of γ-secretase, generating different soluble fragments of APP. However, when the β-secretase acts on the APP first followed by the action of the γ-secretase, the fragments of Αβ-1-40 and Αβ 1-42 are released, the amyloidogenic pathway being launched, this being The last fragment has the greatest capacity for self-aggregation (Hardy J. (1997). Amyloid, the presenilins and Alzheimer's disease. Trends Neurosci. 20 (4): 154-9).

 The presence of Αβ extracellular plaques is a central fact in the neuropathology of AD. The β-amyloid theory (Cummings JL (2004). Alzheimer's disease. N Engl J Med. 351 (1): 56-67), is based on the fact that the aggregates of agregβ are the triggering factor of a multitude of neurotoxic pathways between which may include exitotoxicity, alterations in calcium homeostasis, mass production of free radicals and neuroinflammatory processes. On the other hand, there are several studies that suggest that small peptide oligomers may be the toxic form (Roher AE, Chaney MO, Kuo YM, Webster SD, Stine WB, Haverkamp LJ, Woods AS, Cotter RJ, Tuohy J.., Krafft GA, Bonnell BS, Emmerling MR (1996). Morphology and toxicity of Abeta- (1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease. J. Biol. Chem. 271 (34): 20631-5; Lambert JC, Pasquier F., Cottel D., Frigard B., Amouyel P., Chartier-Harlin MC (1998) A new polymorphism in the APOE promoter associated with risk of developing Alzheimer's disease Hum. Mol. Genet. 7 (3 ): 533-40).

Several studies have shown that PS are found in brains with AD, as well as in normal senile controls whenever they are elderly, suggesting that such plaques could be markers of senility rather than dementia (Maccioni RB, Farias GA, Red . LE, Sekler MA and Kuljis RO (2008) What Have We Learned from the tau hypothesis in: Hypotheses and Research Milestones in Alzheimer ^'s Disease (RB Maccioni & G. Perry, eds.) Springer-Verlag, New York-Heidelberg. ). Accordingly, the formation of amyloid components is common in normal aging, and thus Αβ possibly precedes the formation of NFTs; but the presence of both key cellular events in AD is necessary, to lead in a complementary way to the loss of the activity of affected neurons (Maccioni, RB, Barbeito L., and Muñoz JP (2001). The molecular basis of Alzheimer ^'s disease and other neurodegenerative disorders. Arch. Medical Research. 32: 367-381).

 The tau protein is a protein that is normally associated with microtubules and plays an important role in the assembly of these, such as the stabilization of microtubules against dynamic instability and the union of microtubules with other cytoskeleton filaments (Maccioni RB and Cambiazo V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev. 75 (4): 835-864; Maccioni, RB, Barbeito L, and Muñoz JP (2001). bases of Alzheimer's disease and other neurodegenerative disorders. Arch. Medical Research. 32: 367-381). The tau protein belongs to the family of MAPs or Microtubule Associated Proteins. In humans, this is found almost exclusively in neurons (Maccioni RB and Arechaga J. (1987). "The Cytoskeleton in Cell Differentiation and Development." Oxford University Press, UK 367 pp; Maccioni RB and Cambiazo V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly Physiol Rev. 75 (4): 835-864) and is presented in 6 isoforms, which derive from the expression of a single gene. This gene is found on the long arm of chromosome 17, at position 21 (17q21) and contains 13 exons, which by an alternative splicing process generate 6 molecular isoforms, which have between 352 and 441 amino acids (Goedert M . (2004). Tau protein and neurodegeneration. Seminars in Cell & Developmental Biology. 15: 45-49).

The scheme proposed in Goedert M. (2004). Tau protein and neurodegeneration. Seminars in Cell & Developmental Biology. 15: 45-49, synthesizes information about the structure of the tau gene, and its expression by alternative splicing in six isoforms of the human brain. As a result of this process, 6 isoforms are generated ranging from 45 kDa to 65 kDa, which are differentially expressed during development and can also be distributed in different neuronal subpopulations (Kosik KS, Orecchio LD, Bakalis S., Nevé RL (1989) Developmentally regulated expression of specific tau sequences Neuron 2 (4): 1389-97).

The N-terminal region of the tau protein is of variable length, depending on whether or not the isoform is present, exons 2 and / or 3. This region is known as the projection domain, because once the tau protein interacts with the microtubules, it is projected from them to the outside. Through this projection tau is able to interact with other elements of the cytoskeleton such as actin or spectrin filaments (Cross D., Vial C. and Maccioni RB (1993). A iau-like protein interacts with stress fibers and microtubules inhuman and rodent cell lines J. Cell Sci. 105: 51-60; Farias GA, Muñoz JP, Garrido J., Maccioni RB Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies. (2002) J. Cell Biochem. 85: 315-324; Carlier MF, Simón C, Cassoly R., Pradel LA (1984). Interaction between microtubule-associated protein tau and spectrin. Biochimie. 66: 305-311) or interact with the plasma membrane (Brandt R. , Leger J., Lee G. (1995). Interaction of tau with neural plasma membrane mediated by tau's aminoterminal projection domain. J Cell Biol. 131: 1327-1340; Hernández P., Lee G., Sjoberg M. and Maccioni RB (2008). "Tau phosphorylation by cdk5 and Fyn in response to amyloid peptide Αβ25-35: involvement of lipid rafts". J. A lz. Dis. (In press)

On the other hand, in the C-terminal domain, the domains of the "tandem" repetitive sequences, involved in microtubule binding, are located (Maccioni RB and Arechaga J. (1987). "The Cytoskeleton in Cell Differentiation and Development" Oxford University Press, UK 367 pp; Maccioni RB and Cambiazo V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev. 75 (4): 835-864). These have 3 or 4 segments of 18 amino acids each, highly conserved, which are separated by regions of about 13 amino acids (Maccioni RB, Vera JC, Dominguez J., Ávila J. (1989). A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau Arch Biochem Biophys. 275 (2): 568-79). This binding domain (Maccioni RB, Rivas CI, Vera JC (1988). Differential interaction of synthetic peptides from the carboxy-terminal regulatory domain of tubulin with microtubule-associated proteins. EMBO J. 7 (7): 1957-63) is the person in charge of the assembly and stabilization of the microtubules (Mandelkow EM, Biernat J., Drewes G., Gustke N., Trinczek B., Mandelkow E. (1995). Tau domains, phosphorylation, and interactions with microtubules. Neurobiol Aging. 16 : 355-362). In addition, its association with other proteins such as G-actin has been demonstrated. Maccioni et al., 1989 schematizes the general structure of the major isoform of the tau protein, h 67 (2N4R) of 441 amino acids.

Although the tau protein is capable of undergoing a series of post-translational modifications (Pevalova M., Filipcik P., Novak M., Avila J., Iqbal K. (2006). Post-translational modifications of tau protein. Bratisl Lek Listy. 107 (9-10): 346-53), this phosphorylation plays a particularly important role in the regulation of its activity. The major isoform of tau, has 79 serines or threonines that act as potential phosphorylation sites (Lovestone S., Reynolds CH (1997). The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative process. Neuroso. 78: 309-324 ). Due to this, the combined activity of different protein kinases and phosphatase, which can act on the different isoforms of tau, can generate a high number of structural states in this protein containing different levels of phosphorylation in each of these residues and in each isoform. . Thus, the balance between phosphorylation mechanisms by protein kinases, and dephosphorylation by protein phosphatases, finally modulates a structural state of tau that in short defines its level of activity (Mandelkow EM, Biernat J., Drewes G., Gustke N. , Trinczek B., Mandelkow E. (1995). Tau domains, phosphorylation, and interactions with microtubules. Neurobiol Aging. 16: 355-362; Lovestone S., Reynolds CH (1997). The phosphorylation of tau: a critical stage in neurodevelopment and neurodegenerative process. Neurosc. 78: 309-324). Among the kinase proteins that phosphorylate tau at the cellular level are calmodulin kinase, p38 protein, and Gsk3b enzymes (Rankin CA, Sun Q. and Gamblin TC (2007). Tau phosphorilation by GSK3P promoting tangles-like filament morphology. Molecular Neurodegeneration. 2:12) and cdk5 (also called TPK II), the latter involved in the control of neurogenesis processes commanded by the activity of tau (see Table 1) (Maccioni, RB, Barbeito L, and Muñoz JP ( .. 2001) The molecular basis of Alzheimer ^'s disease and other neurodegenerative disorders Arch Medical Research 32:... 367-381; Reynolds CH, Betts JC, Blackstock WP, Nebreda AR, Andenson BH (2000) Phosphorylation sites on tau Identifies by nanoelectrospay mass spectrometry Differences in vitro between the Mitogen Activated Protein Kinase ERK2. c-Jun N Terminal Kinase and p-38, and Glycogen Synthase Kinase-3b. J. Neuroch. 74: 1587-1595).

It is interesting to note that a protein such as tau, so crucial to define the polarity of neurons and transport processes, generation of growth cones in axonal development, etc. (Kosik KS, Orecchio LD, Bakalis S., Nevé RL (1989). Developmentally regulated expression of specific tau sequences. Neuron. 2 (4): 1389-97; Ferreira A., Cáceres A. (1991). Estrogen-enhanced neurite growth: evidence for a selective induction of Tau and stable microtubules J. Neurosci. 11 (2): 392-400) needs to be modulated by very fine mechanisms for it to fulfill its function in a controlled manner. Thus, important changes in this regulation can modify its ability to bind to microtubules or other elements of the cytoskeleton, or contribute to generating pathological conditions such as tau hyperphosphorylations, and self-aggregation that make it a protein with neurotoxic actions, such as observed in the degeneration of the Alzheimer type (Sánchez MP, Álvarez-Tallada V., Ávila J. (2001). The tau protein in neurodegenerative diseases. Taupatías. Revista de Neurología. 33 (2): 169-177). Unlike other proteins, the tau protein is resistant to temperature and acidic conditions, this allows the process of separation of MAPs and other thermolabile proteins such as tubulin to be carried out. It is also possible to separate it from those that are denaturated in the presence of acids (Farias GA, Muñoz JP, Garrido J., Maccioni RB Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies. (2002). J. Cell Biochem 85: 315-324).

 García T., Jay D. phosphorylation of tau and Alzheimer disease. (2004). Gad Med Mex. 3: 329-33, specifically in Table 1, shows the phosphorylation sites of tau, and the enzymes involved in these processes, from which it can be concluded that various kinases affect phosphorylation of tau at multiple sites, most of them located towards the C-terminal end.

During the pathogenesis process of AD, tau begins to irreversibly hyperphosphorylate at multiple sites (García T., Jay D. Phosphorylation of tau and Alzheimer disease. (2004). Gad Med Mex. 3: 329-33, specifically table 1) , and is integrated into filamentous anomalous structures called paired helical filaments (PHFs), to finally give rise to NFTs, thus losing their key physiological functions such as the definition of neuronal polarity and the control of microtubule-mediated axonal transport (Maccioni RB and Cambiazo V. (1995) Role of microtubule-associated proteins in the control of microtubule assembly Physiol Rev. 75 (4): 835-864). The hyperphosphorylation of tau is the result of the imbalance of the action of different kinases and phosphatases, resulting in a hyperphosphorylated protein that has compromised its normal function and results in neuronal damage (Buée L, Bassiére T., Buée-Scherrer V. (2000). Tau protein isoforms, phosphorylation and role in neurodegenerative disorders. Brain Res. 33: 95-130). Although still under study, it is currently accepted that Αβ oligomers would be, among others, a trigger factor for changes that lead to altering neuronal signaling mechanisms (Roher AE, Chaney MO, Kuo YM, Webster SD, Stine WB, Haverkamp LJ , Woods AS, Cotter RJ, Tuohy JM, Krafft GA, Bonnell BS, Emmerling MR (1996) .orphology and toxicity of Abeta- (1-42) dimer derived from neuritic and vascular amyloid deposits of Alzheimer's disease. J. Biol. Chem 271 (34): 20631-5; Lambert JC, Pasquier F., Cottel D., Frigard B., Amouyel P., Chartier-Harlin MC (1998) A new polymorphism in the APOE promoter associated with risk of developing Alzheimer's disease Hum. Mol. Genet. 7 (3): 533-40) and which lead to tau hyperphosphorylations. This would be the case, an early event in the progression towards the pathogenesis of AD, however the only correlation established between the intensity of the disease and the pathological lesions is presented with the neurofibrillar clews (Maccioni, RB, Barbeito L, and Muñoz JP (2001) The molecular basis of Alzheimer ^'s disease and other neurodegenerative disorders Arch Medical Research 32:.... 367-381).

The cytoskeleton of eukaryotic cells is the cellular structure responsible for neuronal morphology. It is composed of microtubules, actin microfilaments and intermediate filaments (Maccioni RB and Cambiazo V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev. 75 (4): 835-864; Ávila J. , Lucas JJ, Pérez M. and Hernández F. (2004). Role of Tau Protein Both Physiological and Pathological Conditions. Rev. Physiol. 84: 361-384). These polymers they coexist in an interlaced way through a kind of dynamic macromolecular interactions, which determine the organization of this network, which extends in the broad domain of the cytoplasm until interacting with the cell membrane, the nucleus and cellular organelles such as centrosomes, mitochondria, lysosomes , etc. (Maccioni RB and Arechaga J. (1987). "The Cytoskeleton in Cell Differentiation and Development." Oxford University Press, UK 367 pp; Maccioni RB and Cambiazo V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly Physiol Rev. 75 (4): 835-864).

 Microtubules are essential components of the cytoskeleton, responsible for the formation and maintenance of axons, dendrites and specific contacts. MAP proteins contribute to the dynamism and stability of microtubules. Among them is the MAP1A, MAP1 B, MAP2 protein and finally the tau protein (Maccioni RB and Cambiazo V. (1995). Role of microtubule-associated proteins in the control of microtubule assembly. Physiol Rev. 75 (4): 835- 864; Ávila J., Lucas JJ, Pérez M. and Hernández F. (2004). Role of Tau Protein Both Physiological and Pathological Conditions. Rev. Physiol. 84: 361-384) The tau protein is important in the maintenance of neuron polarity! and in the stabilization of a certain architecture in the differentiated neuron. In addition, it has been shown that tau activity is key to the morphogenesis of growth cones in brain neurons, whose structure also involves local actin filament networks (Paglini G., Pigino G., Kunda P., Morfini G., Maccioni R., Quiroga S., Ferreira A., Cáceres A. (1998). Evidence for the participation of the neuron-specific CDK5 activator P35 during laminin-enhanced axonal growth. J. Neurosci. 18 (23): 9858-9869). A decisive role is also proposed in promoting axonal growth (Black MM, Slaughter T., Moshiach S., Obrocka M. and Fischer I. (1996). Tau is enriched on dynamic microtubules in the distal region of growing axons. J. Neuroscience. 16: 3601-3619). On the other hand, MAP-2 would be related rather to the generation of short processes in the neuron.

Quinolines are heterocyclic aromatic organic compounds, which consist of 2 bonded benzene rings, where it is replaced by an atom of N. Quinolines They have applications both industrially and clinically. It should be noted that mainly at the clinical level some quinoline derivatives have been found that are active as anesthetic, antitumor and antimalarial drugs (Campbell SF, Hardstone JD, Palmer MJ (1988). 2, 4-Diamino-6,7-dimethoxyquinoline derivatives as alpha 1- adrenoceptor antagonists and antihypertensive agents. J Med Chem. 31 (5): 1031-5).

 Especially, the quinolines used as antimalarials owe their origin to quinine, a substance used as an antipyretic for hundreds of years. Quinine, however, has drawbacks associated with its toxicity and its administration, so related substances such as chloroquine have been synthesized, which is a quinoline that was developed during World War II and is now the weapon principal used against human malaria (Ridley R. (2002). Medical need, scientific opportunity and the drive for antimalarial drugs. Nature 415: 686-93). According to their effectiveness against the various stages through which the life cycle of plasmodium passes, antimalarials of the quinoline type are classified as "erythrocyte schizonticides", which act in the asexual erythrocyte stages of the parasites interrupting erythrocyte schizogonia, ending thus with the infection and finally eliminating parasites from the body (Baird JK, Basri H., Subianto B., Fryauff DJ, McEIroy PD, Leksana B., Richie TL, Masbar S., Wignall FS, Hoffman SL (1995). of chloroquine resistant Plasmodium vivax with chloroquine and primaquine of halofantrine. J Infeci Dis. 171: 1678-82). Due to these uses of quinolines, preclinical and clinical phase studies have been accepted, so their use in humans is approved.

Particularly, US Pat. No. 7,117,830 and publication USNo.2005 // 009865 propose the use of certain quinoline derivatives in the diagnosis of diseases related to the accumulation of tau protein. In particular, these documents disclose the compounds: a) 4- [2- (2-bencoimidazolyl) ethenyl] -N, N-diethylbenzenamine (BF-126), 2 - [(4- methylamino) phenyl] quinoline (BF-158) and 2- (4-aminophenyl) quinoline (BF-170) and other phenyl compounds derived from quinoline, which are used as in vivo imanology for tau pathology in Alzheimer's disease. In addition, they demonstrate low affinity for beta-amyloid fibers. and high affinity for tau neurofibrillar obillos, as well as other characteristic effects of Alzheimer's disease.

 Publication WO 2008/049047 discloses quinoline derivatives useful in the treatment of diseases related to the liver X receptor, such as acute coronary syndrome, Alzheimer's, diabetes and atherosclerosis. However, the radical R1 of said document always corresponds to an alkyl derivative, however in the present invention the corresponding radical R2 is a phenyl radical.

 EP 1574500 (A1) discloses structurally different quinoline derivatives to the quinoline compounds of the present invention, which can also be used for diagnostic images of diseases in which tau protein accumulates and compositions and kits comprising them. In addition, it teaches a method for staining neurofibrillar tangles in brain samples and a pharmaceutical composition for the treatment and / or prophylaxis of a disease in which the beta sheet structure is the cause or possible cause of the disease.

WO 2009/097401 and WO 2009/097278 disclose compounds derived from 2-amino-quinoline and 6-substituted-thio-2-amino-quinoline, structurally different from the quinoline compounds of the present invention, those that inhibit β- secretase, known as the β-site amyloid cleavage enzyme, BACE, BACE 1, Asp2 or mempasin2, pharmaceutical compositions containing them and their use in the treatment of Alzheimer's disease, senility, dementia and mild cognitive deterioration.

 WO 2009/133692 discloses quinoline-derived compounds, structurally different from the quinoline compounds of the present invention, useful in the treatment and / or prophylaxis of atherosclerosis such as atherosclerosis and arteriosclerosis associated with diabetes, dyslipidemia, hypercholesterolemia, lipid-related diseases , inflammatory diseases induced by an inflammatory cytokine, skin diseases such as allergic skin diseases, diabetes or Alzheimer's disease, senility, dementia and mild cognitive deterioration.

Thus, after failed attempts with drugs that disassemble senile amyloid plaques, much of the worldwide efforts are being directed towards smaller aggregates of tau and the same neurofibrillar clews (NFTs), the present invention provides quinoline compounds that inhibit formation and at the same time disaggregate NFTs, and therefore, would be useful in the treatment and prophylaxis of Alzheimer's disease.

BRIEF DESCRIPTION OF THE FIGURES

Fig. 1: Illustrates the tau purification procedure developed in the invention. Particularly it is a synthesis of the main stages in the purification, the precipitation of the supernatant after the 3rd centrifugation is carried out with ammonium sulfate at 75% saturation.

Fig. 2: Illustrates the structure of astemizole, benzimidazole used in binding and displacement assays and its ³ H-AST radioactive isotope, to evaluate the interaction of quinolines with the tau protein system.

Fig. 3: Schematic illustration of the procedure used for Scatchard assays and displacement of ³ H-AST with quinolines in vitro, where the tubes are incubated for 4 hours at 25 ° C with slight agitation, drying is carried out on filter paper and it is added in vials with 1 ml_ of scintillation liquid, and the last incubation is performed for 10 minutes in the dark.

 Fig. 4a: Illustrates the fluorescence pattern for THQ 55 (see table 1 below) excited at 290 nm and emission at 380 and 480 nm with a 2.0 slit.

 Fig. 4b: Illustrates the fluorescence pattern for THQ 55 (see table 1 below) excited at 290 nm and emission at 380 and 480 nm, with a "slit" of 5.0.

Fig. 5: Illustrates the "Dotblots" of bovine brain tau. Characterization of the purity of the tau protein. The upper part of the figure corresponds to the supernatant obtained from the precipitation with ammonium sulfate and the lower part to the precipitate of the precipitation with ammonium sulfate. A) dotbiot for hTau. B) dotbiot for bTau, where supernatant 1 is the supernatant of precipitation with ammonium sulfate, and precipitate 1 is dialyzed precipitate from saturation with ammonium sulfate.

Fig. 6: Illustrates the purification procedure of tau protein. A) SDS-PAGE. It shows the characterization of bovine tau protein (bTau) obtained from purification with ammonium sulfate. Lane 1: All Blue molecular weight standard (catalog # 161-0373 BIO-RAD); Lanes 2 and 3: 6.0 μί of precipitation supernatant with ammonium sulfate; Lanes 4 and 5: 6.0 μΙ_ bovine tau (bTau) 3.36 mg / mL (used as a positive control); Lanes 6 and 7: 6.0 μΙ_ bTau 10.0 mg / mL. B) Western blot Shows the different tau isoforms obtained in the purification of this protein. The presence of protein isoforms, which are between 55 kDa and 65 kDa, was determined in both the purification of bTau (lanes 2 and 3) and in bTau (lanes 6 and 7). Lane 1: All Blue molecular weight standard (catalog # 161-0373 BIO-RAD); Lanes 2 and 3: 6.0 μΙ_ of bovine tau (bTau) 10.0 mg / mL; Lanes 4 and 5: 6.0 pL bovine tau (bTau) 3.36 mg / mL; Lane 6: 6.0 pL bTau 1.3 mg / mL; Lane 7: 6.0 pL bTau 0.65 mg / mL.

 Fig. 7: Illustrates studies of the morphology of tau filaments in the presence and absence of quinolines. A) Electron micrograph of negative controls (tau without heparin and without quinolines). B) Micrograph of tau filaments obtained with heparin 200 pg / mL, without treatment with quinolines (positive control). The insert corresponds to a magnification of 30,000X. C) Electron micrograph of the incubation of tau with heparin and THQ 55 (see table 1 below). D) Electron micrograph of the incubation of tau with heparin and THQ 4S (see table 1 below). E) Electron micrograph of purified PHFs from human brains through affinity columns. F) PHFs incubated with THQ 55 (see table 1 below).

Fig. 8: Illustrates that quinolines change the structure of the filaments product of the tau aggregate. A) The number of filaments per field after treatment with the quinolines THQ4S and THQ55 is plotted (see table 1 below); B) Length of the Filaments (pm) after these treatments; and C) Width of the filaments obtained (nm). Aggregation studies showed that these quinolines bind to the regulatory region of the C-terminal of tau and prevent their polymerization and affect the structure, (bar 1: polymerization control; bar 2 THQ 4S (see table 1 below); bar 3 THQ 55 (see table 1 below)). Fig. 9: Illustrates the aggregation of the amyloid peptide in the presence and absence of quinolines. A) Electron micrograph of the controls obtained for aggregates of Αβ 1-42 (Negative Control). B) Polymerization of Αβ-42 by stirring at 37 ° C for 7 days (positive control). C) Electron micrograph of incubation of Αβ 1-42 in the presence of THQ 4S (see table 1 below). D) Electron micrograph of Αβ 1-42 incubation in presence of THQ 55 (see table 1 below). All samples were visualized at 15,000X amplification.

 Fig. 10: Illustrates that quinolines slightly change the structure of the filaments of Αβ 1- 42. A) Length of the filaments (μηι) after treatments with the quinolines THQ4S (see table 1 below) and THQ55 (bars 2 and 3) (see table 1 below); Bar 1 indicates the polymerization of the control without quinoline. B) Number of filaments per field after treatment with the quinolines THQ4S (see table 1 below) and THQ55 (bars 2 and 3) (see table 1 below); Bar 1 indicates the polymerization of the control without quinoline.

Fig. 11. Illustrates the turbidimetric study for the aggregation of tau in the absence and presence of quinolines at different concentrations. The curves are colored for different situations: control + (green); control - (red); sample under study plus THQ55 (see table 1 below) at the concentration of 1.0 μΜ (q1 in yellow); sample under study plus THQ55 (see table 1 below) at the concentration of 10 μΜ (q10 in blue); sample of study plus THQ55 (see table 1 below) at the concentration of 50 μΜ (q50 in light blue); shows in study more Astemizole at the concentration of 10 μΜ (Ast 10 in coffee).

Fig. 12. Scatchard chart for ³ H-AST. These saturation tests performed with tau filaments induced with heparin indicate that ³ H-AST binds with great affinity to tau.

Fig. 13: Illustrates the structure of the MN423 monoclonal antibody (light blue ribbon) with the structural core of the tau C-terminal involved in the assembly of the PHFs, corresponding to the pentapeptide ³⁸⁷ DHGAE ³⁹¹ (colored spheres).

 Figure 14. Illustrates the predictive bioinformatic analysis for 6 of the preferential configurations of the interaction of a pentapeptide corresponding to the C-terminal of PHFtau, with the THQ 4S (see table 1 below), modeled by the Autodock ® program. The white arrows indicate the position of the quinoline N.

DESCRIPTION OF THE INVENTION

According to the data provided above, and in the context that the deposits of NFTs in neurons can lead to a gradual neurodegenerative process and about On the basis that these quinolines have a certain affinity for the tau protein (Okamura N., Suemoto T., Furumoto S., Suzuki M., Shimadzu H., Akatsu H., Yamamoto T., Fujiwara H., Nemoto M., Maruyama M., Arai H., Yanai K., Sawada T., Kudo Y. (2005). Quinoline and bencimidazole derivatives: candidate probes for in vivo imaging of tau pathology in Alzheimer's disease. J. Neurosci. 25: 10857-10862) , the inventors directed a search for ligand molecules binding to the tau protein, and especially to the polymerized tau of the Alzheimer type, as potential blockers of hyperphosphorylated tau aggregation before the formation of the NFTs, assessing the ability of a family of quinolines of clinical relevance and its derivatives (THQs) (see Table 1 below), to inhibit the aggregation of tau protein in the form of PHFs produced in vitro, in a possible therapeutic route of AD.

 Table 1: Quinoline derivatives used for evaluation in the disaggregation of tau protein:

 1) 2- (4-methylphenyl), 6- (0-methyl) quinoline (THQ-3S)

 2) 2- (4-methylphenyl), 6- (methyl) quinoline (THQ-4S);

 3) 2- (4-aminophenyl), 6- (methyl) quinoline (THQ-55)

4) 2- (4-aminophenyl) quinoline (THQ-56); 5) 2- (4-methylphenyl), 8- (bromine) quinoline (THQ-9S); Y

 6) 2- (5-methylphenyl), 6- (methyl) quinoline (THQ-12S)

 The aforementioned ligand molecules that interact with the brain tau protein, and that can affect the self-aggregation processes of this hyperphosphorylated protein under pathological conditions, were evaluated with a view to biomedical applications in the domain of Alzheimer's disease research.

 In order to achieve the above objective, cerebral tau and tau protein from paired helical filaments (PHFs) were isolated and characterized from human brains and bovine brain tau which were induced to polymerize with heparin (Mandelkow EM, andelkow E (1993) Tau as a marker for Alzheimer's disease Trends Biochem Sci. 18 (12): 480-3):

 The octanol / water partition coefficient was determined and experimentally correlated liposolubility with the ability of the quinolines to cross the blood-brain barrier (BHE);

Helical filaments of the tau protein were produced and characterized from the isolated and recombinant protein. Alternatively, amyloid aggregates were produced and characterized, from the recombinant peptide Αβ 1-42;

 The interaction of the selected quinolines with the tau protein and its aggregate forms was studied and characterized;

Theoretical studies of computational "docking" were carried out to determine possible interaction sites of the compounds tested within the protein structure.

For the development of the aforementioned relationships, recently slaughtered bovine brains and / or post-mortem human brains stored at -80 ° C, from Alzheimer's patients, were used.

Human PHFs were obtained, which were purified from human brains through affinity columns.

Quinolines of biomedical importance in the context of this invention aimed at the treatment of Alzheimer's disease, as described in Table 1 above, were obtained by organic synthesis. Quinoline samples were prepared at a concentration of 1.0 mg / mL in methanol and serial dilutions were made at different concentrations, at which their fluorescence was determined with an excitation wavelength at 290 nm and one emission between 260 and 500 nm.

The partition coefficient (log P ₀ / w) was determined as described by Takacs-Novak K., Nagy P., Jozan M., Orfi L., Dunn WJ 3rd, Szasz G. (1992). Relationship between partitioning properties and (calculated) molecular surface. SPR investigation of midazoquinazoloneb derivatives. Pharm Hung Act. 62 (1 -2): 55-64, in which 2 phases are used, an aqueous phase corresponding to a PBS buffer solution 0.1X pH 7.4, which is saturated with an n-octanol solution and corresponds to the organic phase.

 Stock solutions of 1.0 mg / mL of each quinoline were prepared under study in PBS buffer 0.1X pH 7.4 saturated in octanol. (THQ 9S and THQ 12S quinoline samples (see table 1 above) were sonicated for a time not exceeding 30 seconds, due to their viscous nature). From these solutions, dilutions were made for a concentration range of 1.0 mg / mL and 100 µg / mL to determine their maximum absorbance in the UV-Visible region.

 Then the tested quinolines were dissolved in PBS 0.1 X pH 7.4 saturated in n-octanol and stirred for 30 min. at room temperature. Subsequently, this emulsion was allowed to stand for 48 h and a centrifugation was performed at 3,000 rpm for 10 minutes to separate both phases. Both the octanol / water partition coefficient and Log P were determined by the absorbance difference in the UV-Visible region of each compound by equation 1:

 Eq. 1: [THQ in octanol] = [initial THQ] - [THQ buffer]

Where:

[THQ buffer] = concentration determined by the absorbance of each sample

Equation 2 determines the concentrations for initial THQ:

 Ec. 2: [initial THQ] = 100 uL x [THQ concentration in methanoll

10,000 ML Subsequently, the quotient P between both phases was determined as described in equation 3:

 Ec. 3: P = í THQ in octanol 1

 [THQ buffer]

Finally the partition coefficient was determined by Log P.

 For this test, 2 controls were used, Clidinium bromide corresponding to a quaternary amine that is not liposoluble, and sodium thiopental, which corresponds to a very fat-soluble barbiturate anesthetic. Each point of the trial was done in triplicate. The tau protein was obtained essentially following the method of Farías (Farías G.A., Vial C, and Maccioni R.B. (1992). Specific macromolecular interactions between tau and the microtubule system. Molecule and Cellular Biochemistry. 112: 81-88) with minor modifications. Recently slaughtered bovine brains and / or post-mortem human brains stored at -80 ° C, were cleaned of meninges, blood vessels and superficial blood; and are processed according to the protocol of repetitive cycles of assembly and disassembly, dependent on temperature.

 Regions of the brain such as the temporal and frontal lobe, which are affected in AD, were homogenized at 4 ° C in homogenization buffer (solution A). The homogenate was centrifuged at 42,000g (19,450 rpm using the T647.5 rotor) for 30 min. at 4 ° C; stage in which the supernatant is collected. Then to this supernatant, the necessary components for the polymerization of MT (solution B) were added. This solution was incubated for 1 h at 37 ° C with mild agitation. The most viscous liquid obtained is divided into the centrifuge tubes. Subsequently, the microtubules formed were rescued from the pellet formed by centrifugation at 42,000g for 30 min at 37 ° C. This microtubule pellet was subsequently treated with the microtubule homogenization buffer (solution C). The volume to be prepared is approximately 60 mL (3 volumes of solution for each volume of pellet).

It was then homogenized on ice with homogenizer "dounce" for 15 min. This step is critical in tau performance. The suspension obtained was subjected to a water bath at 100 ° C for 5 min. to precipitate tubulin and other contaminants that are thermolabile. Subsequently, this suspension was centrifuged at 42,000g for 30 min. at 4 ° C, at which stage the supernatant was rescued, which was treated with ammonium sulfate at a saturation of 75% overnight at 4 ° C, to precipitate the tau protein (Farias GA, Muñoz JP, Garrido J. , Maccioni RB Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies. (2002) J. Cell Biochem. 85: 315-324). Subsequently, it was centrifuged at 42,000g for 30 min. at 4 ° C where the precipitate is resuspended with solution D and dialyzed to remove salts. The dialysis was performed on dialysis membranes of known pore (12 kDa in size) at 4 ° C and under stirring for 24 h with three changes of 2.5 mM Tris-HCI buffer during the 24 h. The solution obtained was concentrated by a "Centricon ultrafiltration" system in a centrifugation at 2,000g for 30 min. at 4 ° C. Figure 1 summarizes the steps performed in the purification of tau.

Protein concentration was determined by the Bradford method (Bradford MM (1976). A Rapid and Sensitive Method for the Quantitation of Microgram Quantities of Protein Utilizing the Princie of Protein Dye Binding. Analytical Biochem. 72: 248-254) using albumin of bovine serum for the calibration curve.

 After purifying the tau protein and determining its concentration, equal amounts of proteins were loaded into 10% polyacrylamide denaturant gels (Laemmli UK (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature. 227 (5259): 680-5); They were then transferred to a nitrocellulose membrane and this was blocked with 5% skim milk in PBS buffer. Subsequently, the membranes were incubated with a primary antibody (1: 1000 dilution in 1% skim milk in 1X PBS) overnight at 4 ° C. After three washes with PBS-Tween (0.05%) the membranes were incubated with a secondary antibody (1: 1000 dilution in 1% skim milk in 1X PBS) associated with peroxidase. Finally, the detection was performed using a luminescence system and the samples were analyzed on photographic plates.

The following primary antibodies were used for immunodetection: to study the phosphorylation states of the tau protein, the AT8 antibody that recognizes the phosphorylated epitopes Ser ⁰² and Thr ²⁰⁵ in the tau protein was used; Tau 5 that recognizes epitopes, of the tau protein, independent of its phosphorylation status; and RBX anti β-amyloid 1-42, which recognizes the amyloid peptide 42 amino acids in length. All these antibodies were used according to the manufacturers instructions.

PROTEIC AGGREGATION STUDIES.

EFFECT OF THE QUINOLINAS IN THE AGGREGATION OF TAU. STUDIES OF ELECTRONIC MICROSCOPY (ME). The purified and highly concentrated tau protein was induced to polymerize in the form of PHFs in the presence of polyanions (heparin at a concentration of 200 μο / mL) in a final volume of 10 μΙ_. This mixture was incubated at 37 ° C and kept under mild agitation for 7 days. At the same time, another sample containing the tau protein, heparin (200 μg / mL) and the different quinolines under study (THQ 4S and THQ 55 (see table 1 above)) were incubated at a concentration of 10 μΜ and final volume of 10 μΙ_, thus verifying the effect that quinolines have on the polymerization of this protein. In parallel, a study was also carried out using recombinant tau protein at a concentration of 2.0 mg / mL prepared in distilled water. Table 2 summarizes the conditions of this experiment.

 The aggregates thus formed were visualized through electron microscopy by negative staining with 2% uranyl acetate. Finally, a count was made of the number of filaments per field, and the measurement of their length and width, observing an average of 25 fields.

TABLE 2: TAU PROTEIN AGGREGATION STUDIES. As a positive control, a solution containing tau and heparin (without quinoline) was used, and as a negative control tau and water; while the samples under study contained tau, heparin and the quinolines THQ 4S or THQ 55 (see table 1 above) at concentrations of 10 μΜ. The "x" indicates presence and the "-" absence.

EFFECT OF KINOLINES ON THE DISASSEMBLY OF PHFS. STUDIES

I. PHFs with a concentration of 400 Mg / mL were incubated with the quinolines under study at a final concentration of 10 μΜ in a volume of 10 µL at 37 ° C with mild agitation for 7 days. The results were visualized through electron microscopy, by negative staining with 2% uranyl acetate.

 EFFECT OF KINOLINES IN THE AGGREGATION OF THE PEPTIDE Αβ 1-42. STUDIES OF ME. The Αβ 1-42 peptide was dissolved in sterile and filtered 1X pH 7.4 buffer, obtaining a final concentration solution 1.0 mg / mL in a final volume of 100 pL. In order to have a comparison parameter with the tau protein, the Αβ 1- 42 peptide was induced to polymerize in the form of rβ fibrils and aggregates. This solution was incubated at 37 ° C and kept under mild agitation for 7 days (Ward RV, Jennings KH, Jepras R., Neville W., Owen DE, Hawkins J., Christie G., Davis JB, George A., Karran EH, and Howlett DR (2000). Fractionation and characterization of oligomeric, protofibrillar and fibrillar forms of beta-amyloid peptide. Biochem J. 348: 137-144). At the same time, another sample containing the Αβ 1-42 peptide and the different quinolines under study (THQ 4S and THQ 55 (see table 1 above)) were incubated at a concentration of 10 μΜ in a final volume of 100 μί, thus verifying the effect that quinolines have on the aggregation of the Αβ 1-42 peptide. Table 3 summarizes the conditions of this experiment.

The aggregates thus formed were visualized through electron microscopy, by negative staining with 2% uranyl acetate. Finally, a count of fibers per field and length measurement of these, observing an average of 25 fields.

 TABLE 3: STUDIES OF AGGREGATION OF THE PEPTIDE Αβ 1-42. As a positive control, a solution containing the β-amyloid peptide was used at a concentration of 1.0 mg / mL under stirring at 37 ° C, unlike the negative control that also contains the β-amyloid peptide, but is stored at - 80 ° C remaining unpolymerized; while the samples under study contained the β-amyloid peptide and the quinolines THQ 4S and THQ 55 (see table 1 above) a

concentrations of 10 μΜ. The "x" indicates presence and the "-" absence.

PREPARATION OF THE SAMPLES FOR ELECTRONIC MICROSCOPY. Protein aggregates of tau, Αβ1-42 and PHFs, were visualized through electron microscopy by negative staining with uranyl acetate. For this, 6.0 μί of sample (tau aggregates, PHFs or 1-β 1-42) were taken, deposited on a copper grid (pretreated with parlodeon and carbon vapor) and adsorbed for 1 minute at room temperature. The excess sample was removed with filter paper. Subsequently, 6.0 pL of 2% uranyl acetate prepared in double-distilled water was deposited on the grid, which is allowed to dry for 30 seconds and is used for negative staining. Finally, the grids are examined under a microscope.

EFFECT OF THE QUINOLINAS IN THE AGGREGATION OF TAU. TURBIDIMETRY STUDIES. Tau aggregation was monitored through its absorbance at λ 340 nm by UV spectrophotometry. The purified tau protein from bovine brain and highly concentrated (2.0 mg / mL) was resuspended in 0.1M MES buffer pH 7.2 and was induced to polymerize in the form of PHFs in the presence of polyanions (heparin at a concentration of 200 μg / mL) in a final volume of 1.0 ml_. This mixture was incubated at 37 ° C and kept under mild agitation for 7 days. At the same time, another sample containing the tau protein, heparin (200 μg / mL) and the quinoline selected at different concentrations from 1.0 to 50 μΜ and a final volume of 1.0 mL were incubated, verifying in this way the effect that quinolines have on tau aggregation. Table 4 summarizes the conditions of this experiment. It is known that some benzimidazole derivatives do not prevent the aggregation of tau; In this context, a control containing tau (2.0 mg / mL), heparin (200 μg / mL) and Astemizole was used at a concentration of 10 μΜ. As an additional control, the tau protein was replaced with water to measure the absorbance of these Quinolines. Each sample was made in triplicate.

 TABLE 4: TURBIDIMETRIC STUDY OF THE AGGREGATION OF TAU IN THE PRESENCE AND ABSENCE OF QUINOLINES. As a positive control, a solution containing tau and heparin was used, and as a negative control tau and water; while the samples under study contained tau, heparin and quinoline THQ 55 (see table 1 above) at different concentrations between 1.0 and 50 μΜ. The other aggregation control contains tau, heparin and Astemizole (AST) at a concentration of 10 μΜ. The "x" indicates presence and the "-" absence.

Tau + THQ Tau + THQ Tau + THQ Tau + AST

Control + Control - 55 * 1.0 mM 55 * 10 mM 55 * 50 mM 10 mM

 Tau protein

 (2.0 mg / mL) X X X X X X

 Heparin (200

 μ9 / πιί) X - X X X X

 MONTH (0.1 M) X X X X X X

 THQ 55 * - - X X X -

AST - - - - - X

 Water

Distilled - X - - - - * see table 1 above

 EFFECT OF THE QUINOLINAS IN THE AGGREGATION OF TAU. SEDIMENTATION STUDIES. After 7 days of incubation at 37 ° C under mild agitation, sedimentation of tau aggregates was performed in the presence and absence of quinolines. For this purpose, 400 μ- of each of the samples prepared for turbidimetric analysis were taken and centrifuged at 42,000g (18,000 rpm using the AH-650 rotor) at 37 ° C for 25 min. The pellet of each sample obtained from this centrifugation was resuspended in 50 µL · 0.1 M slightly alkalized MES buffer (pH 7.2) and the protein concentration in each was determined. In this procedure and because the sample volume is very small, a nanophotometer was used measuring the concentration of sedimented and resuspended protein at λ280 nm.

QUINOLIN INTERACTION WITH TAU AND DISPLACEMENT TESTS WITH

H ³ -AST IN VITRO. In previous tests of our laboratory and also of other authors (Okamura N., Suemoto T., Furumoto S., Suzuki M., Shimadzu H., Akatsu H., Yamamoto T., Fujiwara H., Nemoto M., Maruyama M ., Arai H., Yanai K., Sawada T., Kudo Y. (2005). Quinoline and bencimidazole derivatives: candidate probes for in vivo imaging of tau pathology in Alzheimer's disease. J. Neurosci. 25: 10857-10862; Red L., Avila M., Chandia M., and Maccioni RB (2007). ¹⁸ F Lansoprazole as PET radiotracer. Chemical and biological studies towards the development of a New PET Radiotracer. International Conference on Clinical PET and Molecular Nuclear Medicine 10-14 November, Bangkok) Kd and specific binding maximums were determined for some benzimidazoles for clinical use, including [0-methyl- ³ H] -astemizole (Figure 2) through the established interaction with the tau protein. With this background, Scatchard and displacement tests were carried out with the aforementioned quinolines.

The scheme in Figure 3 summarizes the procedure used in both experiments.

• A fixed concentration of tau protein (230 nM) and variable concentrations of ³ H-AST between 10 and 120 nM were used for the Scatchard test, leading to a final volume of 20 with 8% ethanol (8% EtOH). The tau protein had a treatment prior to this test, where it was incubated at 37 ° C under mild agitation for 7 days. Subsequently the Tau protein and ³ H-AST were incubated for 4 h at 25 ° C with mild agitation. This solution was then filtered under vacuum for 10 minutes and the filter papers are dried at room temperature, and transferred to a vial with 1.0 mL of scintillation liquid. This is left in darkness and finally the counts per minute (cpm) were measured in a scintillation counter device.

• In the drug displacement test, the same procedure mentioned above was used, except that a fixed concentration of Tau protein and ³ H-AST (230 nM and 52 nM respectively) was used, and variable concentrations of the cold ligand ( THQ 4S and THQ 55 (see table 1 above, separately) between 10 nM and 10 μΜ (as indicated in Table 6) leading to a final volume of 20 μί EtOH 8%. Subsequently tau protein, ³ H- AST and the quinolines were incubated for 4 hours at 25 ° C under mild agitation and the procedure of Figure 3 is continued.

TABLE 5: THQ 4S AND THQ55 QUINOLIN CONCENTRATIONS USED IN THE DISPLACEMENT TEST. The cold ligand (THQ 4S and THQ55 (see table 1 above, separately) was used in varying concentrations between 10 nM and 10 μΜ. ³ H-AST and tau protein were used in fixed concentrations of 52 nM and 230 nM respectively.

crystallized structure of a fragment of the tau protein. From the public database of the "Protein Data Bank" (PDB), the structure of a pentapeptide was found, consisting of a structural core of the PHFs located in the C-terminal. 2V17: A, corresponds to the PDB code of ³⁸⁷ DHGAE ³⁹¹ . With this small fragment the studies of "Docking" were carried out, to obtain an approximation of the algorithms that allow to predict the type of interaction that occurs between the quinolines and the tau protein. STATISTICAL ANALYSIS: The standard deviation calculations were made in the Excel spreadsheet (Microsoft Office XP), which measures the dispersion of the values with respect to the mean (average value).

 DESVEST OBSERVATIONS starts from the hypothesis that the arguments represent the sample of a population. If your data represents the total population, DESVESTP is used to calculate the standard deviation.

 • The standard deviation is calculated using the "non-biased" or "n-1" methods.

 • DESVEST uses the following formula:

 Logical values such as TRUE and FALSE and text are ignored.

FLUORESCENCE TESTS.

 To determine the interaction of these quinolines with the tau protein, and obtain a fluorescence pattern of both molecules (Friedhoff P., Schneider A., Mandelkow E.M., and

Mandelkow E. (1998). Rapid Assembly of Alzheimer-like Paired Helical Filaments from

Microtubule-Associated Protein Tau Monitored by Fluorescence in Solution. Biochemistry

37: 10223-10230), this test was performed, where it was found that these compounds generate a low fluorescence and in a high concentration (greater than 1.0 mg / mL), which implies using a larger amount of protein, aspect limiting by the levels of its production, at the time of performing the binding tests and determining constants such as Kd,

Ki and Bmax.

In this way, it was found that quinoline THQ 55 (see table 1 above), was the compound that yielded the best results. This quinoline was at a concentration of 1.0 mg / mL and was excited at λ 290 nm, showed an emission at λ 350 and 480 nm. The Fluorescence intensity was 80 and 30 of a maximum of 1000 in the first test, and 30 and 478 of a maximum of 1000, in the latter case increasing the "slip'O light opening of the equipment. Fluorescence tests were performed also for the quinolines THQ 4S and THS 12S (see table 1 above) but the fluorescence intensity was very low (less than 10 with a maximum of 1000) even when increasing the opening of the "slip".

 Figures 4a) and 4b) show the results obtained in the determination of fluorescence for THQ 55 (see table 1 above).

 DETERMINATION OF THE OCTANOL / WATER PARTITION COEFFICIENT LOG P.

 According to the working hypothesis, what we have looked for in these molecules is to evaluate if they can reach the brain and exert their effect on the affected areas in people suffering from AD. In this context, the first step was to determine the liposolubility expressed as Log P. The physicochemical and pharmacokinetic characteristics of these drugs (Table 6) indicate that their liposolubility (expressed as Log P) is relatively high.

TABLE 6: MOLECULAR PROPERTIES OF THE FAMILY OF DERIVATIVES OF QUINOLINES USED. Octanol / PBS buffer partition coefficient expressed as Log P; TPSA: total area of polar areas; VM: molecular volume; PM: molecular weight; n ON: hydrogen acceptor atoms; n OH NH: hydrogen donor atoms.

The calculation of total polar surface areas (TPSA) made based on the individual contribution of each of the polar groups of these compounds (according to the Ertl method P., Rohde B., Selzer P. (2000). Fast calculation of molecular polar surface area as a sum of fragment-based contributions and its application to the prediction of drug transport properties. J.Med.Chem. 43: 3714-3717) indicates that these drugs have TPSA values similar to other drugs that have good absorption and penetration of BHE. The analysis of molecular properties, according to the "Rule of Five 5" of Lipinski CA, Lombardo L., Bominy BW, Feeney PJ (1997). Experimental and computational approaches to estímate solubility and permeability in drug díscovery and development settings. Adv Drug Delívery Rev. 23: 4-25, which indicates that these drugs have structures that would favor the penetration of BHE into humans.

PURIFICATION OF THE TAU PROTEIN. To determine the purity of the tau protein, it was characterized by "dotbiots" after its precipitation with ammonium sulfate salts and after dialysis. For the "dotbiots", Tau-5 was used as antibody 1 ^no and mouse antilgG developed in peroxidase-conjugated goat as antibody 2 ^r '° (Figure 5).

Through this technique, it was determined that the total tau protein was obtained in the precipitate with ammonium sulfate salts and not in the supernatant. Using SDS-PAGE and Western Blots, the patterns of tau isoforms present in the precipitate obtained from purification were determined (Figure 6).

Through the MicroBradford technique the concentrations of both bTau and Tau, obtained in the purification were determined. Subsequently a lyophilization was performed, to concentrate each of both hTau and bTau samples, from which concentrations were obtained that were adequate for subsequent tests, with a 12% to 14% yield for tau purification, which is in accordance with the method described by (Farías GA, Vial C, and Maccioni RB (1992). Specific macromolecular interactions between tau and the microtubule system. Molecule and Cellular Biochemistry. 112: 81-88; Farias GA, Muñoz JP , Garrido J., Maccioni RB Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies. (2002) J. Cell Biochem. 85: 315-324). Table 7 indicates the total amount of protein obtained in both processes. TABLE 7: CONCENTRATIONS OF THE TAU PROTEIN OBTAINED IN THE PURIFICATION PROCESS. The concentrations were determined by the "microbradford" technique. After purification the samples were lyophilized to obtain the appropriate concentrations for subsequent tests.

AGGREGATION STUDIES

 PROTETICS:

 EFFECT OF THE QUINOLINAS IN THE AGGREGATION OF HUMAN TAU. STUDIES OF ELECTRONIC MICROSCOPY. After 7 days of protein incubation at 37 ° C with mild agitation, tau aggregates with morphological characteristics similar to PHFs were observed. This was obtained with both the tau isolated from bovine brains and the tau isolated from human brains (Figures 7a) and 7b)).

Based on evidence showing that tau mutated with a deletion, and without its C-terminal domain does not generate structures of the PHF type (Jakes R, Novak M., Davison M., and Wischik M. (1991). Identification of 3 and 4 repeat tau isoforms within the PHF in Alzheimer's Disease. EMBO J. 10: 2725-2729, the aggregation studies that show PHF formation, indicate that the different quinolines would bind to the regulatory region of the C-terminal of tau. the quinolines would prevent its polymerization, also affecting the structure of the filaments (Figure 7c) and 7d)). The results also showed a decrease in both the number of filaments per field, as well as in the length and width in each of the filamentous structures (Figure 8). When these quinolines (at a final concentration of 10 μΜ) were also incubated together with the PHFs purified from human brains, a decrease in the number of aggregates presented by the controls was observed. This result would indicate that in addition to preventing polymerization, these quinolines would disaggregate the PHF structures formed in patients affected by AD (Figures 7e) and 7f)).

 EFFECT OF KINOLINES IN THE AGGREGATION OF THE PEPTIDE Αβ 1-42. STUDIES OF ELECTRONIC MICROSCOPY (ME). After 7 days of incubation at 37 ° C with mild agitation, aggregates of this peptide in the form of amyloid fibers were achieved (Figure 9).

 These aggregation studies showed that quinolines, as in tau aggregation, prevent imerβ1-42 peptide polymerization and also affect the structure of the filaments. The results showed a decrease in the number of filaments per field, but an increase in the length of the fibers (Figure 10). This is an interesting observation that is important to highlight because it differentiates the effects of quinolines on tau aggregates and those of Αβ 1-42. This sedimentation test (Maccioni RB and Seeds NW (1978). Enhancent of tubulin assembly as monitored by a rapid filtration assay. Arch Biochem Biophys. 185 (1): 262-71) further indicates that the total mass of added amyloid would not decrease substantially, unlike tau polymers, in that there is apparently a decrease in the amount of tau monomers that are added. In addition to analyzing the structural changes of tau filaments obtained in the presence and absence of quinolines, it was of interest to quantify the effect of these molecules on said aggregates. For this, sedimentation tests were performed (Maccioni RB, Vera JC, Dominguez J., Ávila J. (1989). A discrete repeated sequence defines a tubulin binding domain on microtubule-associated protein tau. Arch Biochem Biophys. 275 (2) : 568-79) and turbidimetry of tau polymers in the absence and presence of quinolines, analyzing the amount of tau polymerized at different concentrations of the drug.

EFFECT OF THE QUINOLINAS IN THE AGGREGATION OF TAU. TURBIDIMETRY STUDIES.

Inhibition of tau protein aggregation is an important aspect of a potential therapeutic compound for the treatment of AD. The quinoline THQ 55 (see table 1 above) was used for this purpose. This drug showed a great capacity to inhibit tau aggregation, which was verified by measuring Absorbance at λ 340 (Figure 11), in where it is observed that at very low concentrations the drug has a potential effect on aggregation.

 EFFECT OF THE QUINOLINAS IN THE AGGREGATION OF TAU. SEDIMENTATION STUDIES:

After 7 days of incubation at 37 ° C under mild agitation, the concentration of sedimented protein in the presence and absence of quinolines and measured at λ280 nm showed a marked inhibitory effect of THQ 55 (see table 1 above) on the self-aggregation of Tau protein in in vitro assays. This inhibitory effect was even more marked at concentrations of THQ 55 (see table 1 above) above 10 μ. Table 8 shows the concentrations of tau polymers obtained in sedimentation.

TABLE 8: SEDIMENTATION STUDY FOR THE ADDING OF TAU IN ABSENCE AND PRESENCE OF QUINOLINAS. As a positive control a solution containing tau, and heparin was used, and as a negative control only tau and water; while the samples under study contained tau protein, heparin and quinoline THQ 55 (see table 1 above) at different concentrations between 1.0 and 50 μΜ. The other aggregation control contains tau, heparin and Astemizole (AST) at a concentration of 10 μΜ.

 QUINOLIN INTERACTION WITH TAU AND DISPLACEMENT TESTS WITH

3H-AST IN VITRO. Based on the characterization of the íat / -benzimidazoles system, the protein-ligand interaction parameters were studied. The results are shown in Figure 12 and Table 9 and indicate that Astemizol binds with great affinity to tau filaments. This phenomenon is repeated when comparing affinity for tau filaments induced in vitro and for isolated filaments of brains with EA (Red L, Avila M., Chandia M., and Maccioni RB (2007). ¹⁸ F Lansoprazole as PET radiotracer. Chemical and biological studies towards the development of a New PET Radiotracer. International Conference on Clinical PET and Molecular Nuclear Medicine 10-14 November. Bangkok).

In this context, a ³ H-AST displacement test with the quinolines was performed to determine its affinity with tau. The results shown in Figure 13, account for the affinity of quinolines for tau aggregates. It is observed that the compounds THQ 4S and THQ 55 (see table 1 above), have very high Ki values being greater than 10 μΜ, indicating that these drugs do not displace the radioligand.

TABLE 9: COMPARATIVE ANALYSIS OF SATURATION DATA. Kd, Bmax, and Kb / Bmax for ³ H-AST and aggregate forms of tau.

"DOCKING" TESTS. Since tau is a very fibrous protein with a large domain structured as a statistical ball ("random coiled") (Von Bergen M., Barghorn S., Biernat J., Mandelkow EM, Mandelkow E. (2005). Tau aggregation is driven by a transition from random coil to beta sheet structure Biochimica et Biophysica Acta. 1739: 158-166) a complete crystallization of this protein has not been achieved, only segments of it.Tau is not a protein with a regular structure in its entirety Therefore, to date the crystal structure of the whole protein is not known and only the study by Novak M., Wischik CM, Edwards P., Pannell R., Milstein C. (1989). Characterization of the first monoclonal antibody against the pronase resistant core of Alzheimer's PHF. Prg Clin Biol Res. 317: 755-61, describes the only known fragment, which consists of a pentapeptide ³⁸⁷ DHGAE ³⁹¹ located in the C-terminal domain and that is involved in a t assembly regulatory region au to form the PHFs. This region contributes to the reactivity of the MN423 monoclonal antibody (Figure 13) (Sevcik J., Skrabana R., Dvorsky R., Csokova N., Iqbal K., Novak M. (2007). X-ray structure of the PHF core C-terminus: insight into the folding of the intrinsically disordered protein tau in Alzheimer's disease. Lett. 581 (30): 5872-5878). Thus, on this basis, modeling was performed with this tau fragment because it is exposed in the paired filaments of the protein as described by Novak M., Wischik CM, Edwards P., Pannell R., Milstein C. ( 1989). Characterization of the first monoclonal antibody against the pronase resistant core of Alzheimer PHF. Prg Clin Biol Res. 317: 755-61 and Skrabana R., Skrabanova M., Csokova N., Sevcik J., Novak M. (2006). Intrinsically disordered tau protein in Alzheimer's tangles: a coincidence or a rule? Bratisl Lek Listy. 107 (9-10): 354-8.

 To complement this analysis, computerized "docking" studies were carried out with the Autodock III® program using this crystallographic structure known to date for the tau protein or fragments thereof. Docking studies were performed, obtaining an approximation of the algorithms that allow predicting the most stable type of interaction produced between the quinolines and the tau protein. The "docking" between this pentapeptide and the quinoline THQ 4S (see table 1 above), resulted in the most likely interactions (Figure 14) with their respective "docking" energies ranging from -4.5 to -4.47 Kcal / mol.

Table 10 summarizes the results obtained from the bioinformatic "docking" analysis for THQ 4S (see table 1 above) and the pronase resistant fragment PHF-teu- ³⁸⁷ DHGAE ³⁹¹ of which its crystalline structure is well known. Thus, the binding energies described in this table are compared with those obtained by the same program for drugs with high affinity such as the metabotropic glutamate receptor (mGluR) (Yanamala N., Tirupula KC and Klein-Seetharaman J. (2008) Preferential binding of allosteric modulators to active and inactive conformational states of metabotropic glutamate receptors. BMC Bioinformatics. 9 (Suppl 1): S16). The more negative values of binding energies appear to be more thermodynamically favorable if they are related in terms of free energy (AG), indicating that this quinoline is akin to fragment ³⁸⁷ DHGAE ³⁹¹ of tau, suggesting that this fragment might be involved in its union and antiaggregant activity.

TABLE 10: COMPARISON OF "DOCKING" ENERGIES. Energies obtained for quinoline THQ 4S, compared to the docking energies obtained for a high affinity receiver. Energies of

 "Docking"

 Ligand Species Receptor (Kcal / mol) Source

 THQ 4S fragment

of Human tau (see table 1) -4.5 to -4.47 Oral Communication ⁽ '

 Yanamala N., Tirupula K.C. and Klein- Seetharaman J. (2008). Preferential binding of allosteric modulators to active and ¡nactive conformational states of metabotropic glutamate receptors. BMC Bioinformatics. 9 mGluR 1 Human R214127 -7.34 (Suppl 1): S16

 Yanamala N., Tirupula K.C. and Klein- Seetharaman J. (2008). Preferential binding of allosteric modulators to active and ¡nactive conformational states of metabotropic glutamate receptors. BMC Bioinformatics. 9 mGluR 5 Human PEP -7.77 (Suppl 1): S16

As mentioned before, the objectives of this invention were focused on the search for polymerized tau protein binding ligand molecules, as potential blockers of tau aggregation before NFT formation. This is how it was possible to determine the way in which a family of different quinolines interact with the tau protein, and in this way, obtain a biomedical projection within the EA. This would be the first finding described so far, which manages to identify a new family of molecules that bind tau while interfering with their pathological self-assembly on the path to neurodegeneration. The study was carried out essentially in in vitro models, and according to the results obtained, it can be concluded that these quinolines would be serious blocking candidates for tau aggregation in their polymerized form of PHFs.

FLUORESCENCE TESTS. This study was aimed at determining the affinity of quinolines for the tau protein and its aggregates and corroborate what was previously stated by other authors (Rojo L, Avila M., Chandia M., and Maccioni RB (2007). ¹⁸ F Lansoprazole as PET Radiotracer Chemical and biological studies towards the development of a New PET Radiotracer. International Conference on Clinical PET and Molecular Nuclear Medicine 10-14 November. Bangkok; Okamura N., Suemoto T., Furumoto S., Suzuki M., Shimadzu H., Akatsu H., Yamamoto T., Fujiwara H., Nemoto M., Maruyama M., Arai H., Yanai K., Sawada T ., Kudo Y. (2005). Quinoline and bencimidazole derivatives: candidate probes for in vivo imaging of tau pathology in Alzheimer's disease. J. Neurosci. 25: 10857-10862). Fluorescence assays were performed for this purpose, but it was found that the emission pattern of these compounds was very low and it was necessary to use very high drug concentrations to obtain a signal. In this way, and according to the amounts of quinoline needed to obtain a signal, this binding assay could not be considered, because the amount of protein to be used for binding with quinoline was too high.

DETERMINATION OF THE OCTANOL / WATER PARTITION COEFFICIENT LOGP. According to the development of this invention, one of the first properties analyzed was whether these molecules, the quinolines, managed to cross the blood brain barrier and reach the brain. These presented a relatively high fat solubility. The correlation between the liposolubility of quinolines with the ability to cross the blood-brain barrier (BHE) was optimal for 3 of the 6 compounds. The molecular properties analysis found that quinolines have similar properties to other drugs that cross the BHE. The result was positive as shown throughout this description and suggests that these molecules could cross the BHE according to in vitro model studies. In this context, these compounds must meet certain requirements to be of interest in a potential pharmacological application at the neural level: (1) They must be highly lipophilic and have the ability to cross the blood brain barrier (BHE); (2) Act at low concentration and remain short in brain tissue; (3) Interact with the tau protein and its aggregates; (4) Have a very low nonspecific union. Thus, these drugs could have a direct effect on brain regions in people suffering from AD. The quinolines described here fully comply with these requirements, namely they have the property of being highly lipophilic, interact with tau and exert their action in blocking their self-aggregation in pathological polymers, such as PHFs. We believe that this is a remarkable finding, since until today no molecules have been described that meet these properties.

 One of the first trials was to determine the liposolubility expressed as Log P. Our results show that the liposolubility of the compounds THQ 4S, THQ 55 and THQ 12S (see table 1 above) is optimal and makes them serious candidates to cross the BHE. According to other physicochemical and pharmacokinetic characteristics shown in Table 2, it is shown that these quinolines have TPSA values similar to other drugs that have good absorption and penetration of the blood brain barrier. The analysis of molecular properties, according to the "Rule of Five 5" by Lipinski C.A., Lombardo L, Bominy B.W., Feeney P.J. (1997). Experimental and computational approaches to estímate solubilíty and permeabilíty in drug díscovery and development settings. Adv Drug Delivery Rev. 23: 4-25, indicates that these drugs have structures that would favor the penetration of BHE in humans and could exert their action at the brain level. An important fact is that many clinical drugs used as antimalarials have in their structure the core of quinolines. In this context its use in humans is approved and for pharmacological evaluation, preclinical phase studies would be avoided. Based on the above, and according to their molecular properties, THQ 4S and THQ 55 (see table 1 above) were used for biological tests. As an annex, it is known that the permeability of the neuronal membrane also represents an important factor for obtaining images of intracellular aggregates of tau (Small GW, Agdeppa ED, Kepe V., Satyamurthy N., Huang SC, Barrio IR (2002). In vivo brain imaging of tangle burden in humans J. Mol. Neurosci. 19: 323-327), where the high liposolubility of these compounds is an advantage.

PURIFICATION OF THE TAU PROTEIN. As for the purification of the tau protein, this protein is resistant to acidic conditions, so tau was usually purified by precipitation with perchloric acid (Farías GA, Vial C, and Maccioni RB (1992). Specific macromolecular interactions between tau and the microtubule system, Molecule and Cellular Biochemistry 112: 81-88) in one of the last steps of purification. Subsequently, based on the disclosures of Farias GA, Muñoz JP, Garrido J., Maccioni RB Tubulin, actin, and tau protein interactions and the study of their macromolecular assemblies. (2002) J. Cell Biochem. 85: 315-324, the use of ammonium sulfate was introduced as a variant, which allowed tau to be concentrated with a slight increase in its purity. It should be noted that the critical stage in tau purification lies in the correct use of the polymerization and depolymerization cycles as originally described by this laboratory (Maccioni RB, Rivas CI, Vera JC (1988). Differential interaction of synthetic peptides from the carboxyl-terminal regulatory domain of tubulin with microtubule-associated proteins. EMBO J. 7 (7): 1957-63).

PROTEIN AGGREGATION STUDIES. One of the most important trials was to visualize how these drugs block polymerization of the tau protein. The results show a clear inhibition of the tau protein in its aggregate form of PHFs, because the incubation of the quinolines with the protein decreased both the number of filaments per field, and the length and width of these filamentous structures. By way of comparison, the same assay was performed with the human recombinant tau protein, obtaining the same results, but in this protein the number of aggregates was always lower, because the recombinant protein is not phosphorylated and the number of PHFs when incubated With heparin is decreased. The quinolines decreased 5 times the length and 10 times the width of the tau filaments formed in vitro. To verify that these drugs have a greater affinity for the tau protein than for the Αβ aggregates, the same previous tests were performed, finding that the quinolines decreased the number of amyloid aggregates, but increased the length of the fibers. The quinolines decreased the number of fibers but increased their length, suggesting that it would be related to mass redistribution and not an impediment to aggregation. This is an interesting observation that is important to highlight because it differentiates the effects of quinolines on tau aggregates and those of Αβ 1-42, indicating that the total mass of added amyloid would not decrease substantially, but would be redistributed unlike polymers. of tau, in which there is a decrease in the amount of tau monomers that are added, as described below: TURBIDIMETRY AND SEDIMENTATION TESTS. Electron Microscopy data were corroborated by sedimentation tests as well as turbidimetric tests, with a marked inhibitory effect of THQ 55 (see table 1) on self-aggregation of tau protein in in vitro assays. A strong demonstration of the ability of these quinolines to affect tau self-aggregation was obtained by turbidimetric studies, followed by sedimentation tests of tau polymers in the presence of increasing concentrations of quinoline (THQ55, see table 1). Thus, in addition to the fine microscopy studies, the sedimentation and turbidimetric tests allowed us to directly verify the ability of these drugs to inhibit tau aggregation. This inhibitory effect was even more marked at THQ 55 concentrations (see table 1) greater than 10 μΜ (see Figure 10 and Table 8). These results suggest that this quinoline appears as a potential candidate to be a tau antiaggregant, which would be of great relevance as a therapeutic approach towards the control of neurofibrillar clews in Alzheimer's Disease.

On the other hand, it should be noted that many small molecules that have been used as inhibitors of amyloid polymerization in vitro contain aromatic rings in their structure. These inhibitors include Congo Red for pamiloid, and anthraquinone, and porphyrins for tau (Pickhardt M., Gazova Z., von Bergen M., Khilistunova I., Wang Y., Hascher A., Mandelkow EM, Biernat J. and Mandelkow E (2005) Anthraquinones inhibit tau aggregation and disssolve paired helical filaments in vitro and in cells J. Biol. Chem. 280: 3628-3635; Inouye H., Sharma D., Goux WJ and Kirschner DA (2006). Structure of core domain of fibril-forming PHF / tau fragment Biophys J. 90: 1774-1789). Unlike anthraquinones and porphyrins, quinolines are serious candidates for tau antiaggregant therapy, because they cross BHE in addition to blocking the aggregation of tau at low concentration and also its use in patients has already been approved unlike porphyrins and anthraquinones. Previous studies of electron microscopy and X-ray diffraction applied to analogues of the tau protein, showed that only 3 residues that make up a β sheet could be involved in its polymerization in filamentous forms of PHFs (Inouye H., Sharma D., Goux WJ and Kirschner DA (2006). Structure of core domain of fibril-forming PHF / tau fragment Biophys J. 90: 1774-1789). This is in accordance with other studies that indicate that these interactions are established through bridges of H or aromatic residues between small peptides (Gazit E. (2002). A possible role for π-stacking in the self-assembly of amyloids fibrils. FASEB J. 16: 77-83; Makin OS, Atkins E., Sirkoski P., Johanson J. and Serpell LC (2005). Molecular basis for amyloid fibril formation and stability. Proc Nati Acad Sci USA. 102: 315-320) . These types of interactions could be used as a reasonable target to interfere with fiber formation or destroy the fiber once formed. These studies are corroborated by the results obtained in our experiments, given that quinolines correspond to 2 aromatic rings, very similar to naphthalene, but with an N in position 1. Another study by Inouye H., and Kischner DA (1991). Folding and function of the myelin proteins from primary sequence data. J. Neurosc. Res. 28: 1-17 to a tau domain located at the C-terminal end, consisting of a small peptide involved in nucleation and polymerization in PHFs, indicates a possible interaction between tyrosines, suggesting that inhibitors could bind to aromatic residues through interactions Hunter CA, Sanders JKM (1990). The nature of n-dnteractions. J. Am. Chem. Soc. 1 12: 5525-5534). DISPLACEMENT TESTS WITH ³ H-AST IN VITRO. Based on the findings in our laboratory and by other authors that benzimidazole derivatives bind specifically and with great affinity to tau protein and NFTs (Rojo L, Avila M., Chandia M., and Maccioni RB (2007). ¹⁸ F Lansoprazole as PET radiotracer Chemical and biological studies towards the development of a New PET Radiotracer International Conference on Clinical PET and Molecular Nuclear Medicine November 10-14 November Bangkok; Okamura N., Suemoto T., Furumoto S., Suzuki M ., Shimadzu H., Akatsu H., Yamamoto T., Fujiwara H., Nemoto M., Maruyama M., Arai H., Yanai K., Sawada T., Kudo Y. (2005). Quinoline and bencimidazole derivatives: candidate probes for in vivo imaging of tau pathology in Alzheimer's disease (J. Neurosci. 25: 10857-10862), raised the possibility that quinolines could displace this interaction. This, based on its similar structure in the nucleus of the benzene ring and the N in position 1 of the benzimidazole ring, could thus displace the benzimidazole and bind to the aggregates of the tau \ n vitro protein. For H ³ -AST was used for this purpose, and the results show that the inhibition constants (Ki) obtained were very high (greater than 10 μΜ), indicating that these drugs do not displace the radioligand, so the only option to obtain the The affinity of these compounds for the protein would be to use a radiolabeled quinoline, which was not possible to obtain commercially. In sum, this experiment showed that the two quinolines do not have the ability to displace used benzimidazoles, which suggests that because these quinolines have a very high Ki, they have a lower tau affinity than benzimidazole or that the sites The interaction of both molecules with the tau protein are different, an aspect that should be investigated.

"DOCKING" TESTS. In this context, docking tests were also carried out to predict the way quinolines interact with the tau protein. To perform these studies it was necessary to obtain a fragment of the crystallized tau protein. This point is key, because if the crystallized structure of the protein does not exist, it is not possible to do the "docking". Studies conducted by Glabe CG (2004). Conformation dependent antibodies target diseases of protein misfolding. Trends Biochem Sci. 29: 542-547, indicate that the tertiary structure of monoclonal antibodies is an essential tool for the investigation of the pathological assembly mechanisms of intrinsically disordered proteins (IDPs), as is the case of tau. Previous studies carried out by Csokova (Csóková N, Skrabana R, Urbániková L, Kovácech B, Popov A, Sevcík J, Novák M. (2006) .Preparation, crystallization and preliminary X-ray analysis of the Fab fragment of monoclonal antibody MN423, revealing the structural aspects of Alzheimer's paired helical filaments. Protein Pept Lett. 13: 941-4) found that the MN-423 monoclonal antibody specifically binds to a structural core of PHF / tau, corresponding to a 93-95 amino acid sequence of C-terminal tau resistant to pronase. Using this tau sequence (amino acids 306-391), the inventors performed the crystallization of the protein for 3 months, where detailed analysis showed that a ³⁸⁷ DHGAE ³⁹¹ pentapeptide that contributes to antibody reactivity is involved in the assembly of the tau protein in PHF structures, converting the known sequence into beta structure during filament assembly (Sevcik J., Skrabana R., Dvorsky R., Csokova N., Iqbal K., Novak M. (2007). X-ray structure of the PHF core C-terminus: insight into the folding of the intrinsically disordered protein tau in Alzheimer's disease. FEBS Lett. 581 (30): 5872-5878). It is also important to add that this sequence is in an area that is highly conserved within tau, corresponding to the repetitive regions of the C-terminal. Based on the previous information and using this sequence, the "docking" studies were carried out, which represent the first approach based on mathematical models between the interaction of quinolines and tau. Through these tests it was determined that these quinolines, specifically THQ 4S (see table 1 above) have docking energies favorable for the interaction between these compounds and a fragment of the tau protein. THQ 4S (see table 1 above) was chosen for this test due to the advantages of its molecular properties and being more lipophilic compared to THQ 55 (see table 1 above). The "docking" of THQ 55 (see table 1 above) with this tau fragment is an aspect that remains to be investigated, although possibly the interaction energies of THQ 55 (see table 1 above) are more negative than those of THQ 4S (see table 1 above), because THQ 55 (see table 1 above) presents electron donor groups in the substituents, which could influence the resonance of the quinoline aromatic rings and delocalize the electrons leaving the N more negative and favoring the interaction with a cationic protein such as tau. This is further corroborated with an important data obtained from this approach, in which this N of the quinoline (Figure 22, indicated with white arrows) interacts with an O that protrudes at the final end of the peptide, corresponding to a small cavity that forms between the Wing and Glu ("pocket" structures). This nitrogen could be replacing an N of Arg 106 of the specific MNβ MN-423. Although the results obtained are relevant, the binding domain cannot be determined exactly, because the peptide is very small and only gives us an approximation of a possible interaction between tau and the quinolines.

Thus, the objectives of this invention were mainly focused on the search for polymerized tau binding ligand molecules, as potential blockers of tau aggregation before the formation of NFTs. Fine microscopy studies, sedimentation and turbidimetric tests allowed directly checking the ability of these drugs to inhibit tau aggregation.

 On the other hand, these molecules have a relatively high fat solubility, and have similar molecular properties to other drugs that cross the BHE, whereby they can eventually exert an action at the brain level. From these results it is concluded that these quinolines and their derivatives can be used as potential tau aggregation inhibitor drugs, in a possible therapeutic route for the treatment of AD.

Claims

 1. Quinoline derivative of formula

, where R ₂ is 2- (4-aminophenyl) or 2- (4-methylphenyl) and R ₆ is methyl, useful as inhibitors of tau protein aggregation.

2. The quinoline derivative of claim 1, wherein R ₂ is 2- (4-aminophenyl) and R ₆ is methyl.

 3. The quinoline derivative of claim 1 or 2, useful for the treatment of Alzheimer's disease (AD).

 4. Use of a quinoline derivative of formula

where R ₂ is 2- (4-aminophenyl) or 2- (4-methylphenyl) and R ₆ is methyl, to prepare a medicament useful for inhibiting the aggregation of tau protein.

5. The use of claim 4, wherein R ₂ is 2- (4-aminophenyl) and R ₆ is methyl.

 6. The use of claim 4 or 5, to prepare a medicament useful for the treatment of Alzheimer's disease.

 7. Method for inhibiting aggregation of the tau protein, which comprises administering a quinoline derivative of the formula

, where R ₂ is 2- (4-aminophenyl) or 2- (4-methylphenyl) and

R ₆ is methyl.

8. The method of claim 8, wherein R ₂ is 2- (4-aminophenyl) and R ₆ is methyl.

9. Method for the treatment of Alzheimer's disease, which comprises administering a quinoline derivative of the formula

, where R ₂ is 2- (4-aminophenyl) or 2- (4-methylphenyl) and

R ₆ is methyl.

10. The method of claim 9, wherein R ₂ is 2- (4-aminophenyl) and R ₆ is methyl.